Light-triggered CO release from nanoporous nonwovens †

Friedrich Schiller University Jena, Institute (IAAC), Humboldtstr. 8, 07743 Jena, Germa de; Fax: +49 3641 948 102; Tel: +49 3641 9 INNOVENT e.V., Biomaterials Department, Institute of Photonic Technology, Albert-Ein Friedrich Schiller University Jena, Jena Cen 10, 07743 Jena, Germany Institute of Physical Chemistry and Abbe University Jena, Max Wien Platz 1, 07743 Je Center for Sepsis Control and Care, Jena 07747 Jena, Germany † Electronic supplementary information DSC, BET, Hg porosity measurements, assay, and ICP-MS. See DOI: 10.1039/c3tb Cite this: DOI: 10.1039/c3tb21649g


A Introduction
2][3][4] CO relaxes smooth muscles at low concentrations and is involved in wound healing and cardiovascular protection. 5,6Furthermore, HO-decient mice show an increased lethality during polymicrobial sepsis, 7 suggesting that the clearance of heme and/or the production of CO is benecial.With regard to the second point CO attracts particular attention as a potential therapeutic agent.10][11] However, its practical clinical use is currently hampered by the methods to deliver CO precisely and safely to target locations. 4O releasing molecules (CORMs) as carrier systems that release CO only aer directed triggering appear as a convenient alternative to the application of pure CO gas. 1 CORMs can be generated from main or transition metal carbonyl complexes as well as from organic molecules.Recent developments have been summarized in excellent reviews.4,[12][13][14][15][16][17][18] The manifold impact of CO on regulatory processes facilitates broad possibilities for medicinal CORM application. 1 For example, the rutheniumbased tricarbonyl complex CORM-3 has been successfully tested in models of vascular dysfunction, ischemic injury and inammation.19 Poole et al. demonstrated that CORM-3 can enter cells, transfer CO intracellularly and inhibit bacterial growth by complex interactions with the respiratory chain in Pseudomonas aeruginosa and Escherichia coli.20 Importantly, it has been recently shown that CORM-3 derived CO and not postrelease metal fragments (e.g. iactive CORM, iCORM) caused the observed effects on respiration.Administration of CORM-2 (tricarbonyldichloro-ruthenium(II) dimer) in rat aortic smooth muscle cells showed contrasting context-dependent effects on the vessel tone demonstrating the complex actions of CO during muscle relaxation.21 CORM-2, just as CORM-3, has versatile effects on pathogenic bacteria, such as E. coli and Staphylococcus aureus, resulting in rapid cell death. This efect on bactericidal cell viability is lost in the presence of a CO scavenger, revealing that released CO and not metal degradation products are responsible for the antimicrobial action.22 Dimanganese decacarbonyl (CORM-1) can play an important role in vascular control as shown with isolated rat hearts.,24 Light stimulated CORM-1 (ref.23) (just as gaseous CO) is able to activate calcium-dependent potassium channels in smooth muscle cells.21,25,26 Further, CORM-1 derived CO showed a positive effect on renal circulation in rats and anti-inammatory effects were demonstrated in a mesenteric microcirculation model.19 However, the water insoluble photoCORM CORM-1 is much less used in medical experiments compared to its soluble analogues.Previous experiments always required the use of DMSO as a (co)solvent. Onthe other hand CORM-1 is a highly loaded CO storage molecule (10 mole CO per mole CORM).The desired water accessibility of CORM-1 for medical applications can be achieved by a pharmaceutical formulation: CORM-1 is incorporated into a biocompatible support matrix (Fig. 1a).Subsequently, the resulting hybrid material can be attached to the desired environment/tissue.[27][28][29] This novel concept reveals also a possible solution to the metal fragment problem (Fig. 1a): CORM degradation products of water soluble CORMs (or even the CORMs themselves) can cause dramatic side effects within cells.1,21,30 Instead, the leover metal fragments would be kept trapped within the scaffold aer release of the biologically active drug.4,16,27,31 Other groups used immobilization strategies of CORMs to address specic biological targets, novel release mechanisms, and stability issues in physiological medium.[32][33][34][35][36][37][38] To our knowledge, only three examples have been published where Mn 2 (CO) 10 has been introduced into a cyclodextrin, cellulose or polymer matrix, but never with the intention to use the CORM-1 derived CO. 39-41 However, brous non-wovens with NO donors have already been obtained via electrospinning. 27,42,43lectrospun materials are highly desirable, since the hybrid matrices are rapidly obtained from the support material and the embedded substance without covalent attachment strategies.The electrospinning technique allows the formation of materials with complex properties and morphologies (e.g.high surface area) that match specic requirements (e.g.efficient gas exchange). 44,45n terms of nanober morphology, porous instead of smooth bers are advantageous for a variety of medical or industrial applications, such as tissue engineering, drug delivery, catalysis or ltration. 44,46Matrix porosity and the resulting high specic surface area allow the tuning of drug release proles from such materials.In the eld of sensor development a highly porous structure is required to ensure high sensitivity and fast sensor response.Electrospun carbon nanobers have proved their increased sensor ability and sensitivity towards NO and CO gases due to their porous structure. 47n the present work CORM-1 was introduced into a polymeric support material to render the compound accessible to water and biological environments (Fig. 1).With this novel procedure not only a drug container was created, but also the advantageous CO release properties of CORM-1 to generate porous polymeric materials could be used. 31This dual use of CORM-1 provides a new way for the production of porous electrospun bers 44 and their use as CO releasing materials (CORMAs). 4,16

B Results and discussion
Synthesis and characterization of CORMA-1-PLA The water insoluble CO-releasing molecule dimanganese decacarbonyl (CORM-1) has been non-covalently incorporated into a nanobrous poly(L-lactide-co-D/L-lactide) (70 : 30, PLA) nonwoven via the electrospinning technique. 44,45,4829]49 For the preparation of the hybrid material rst PLA was dissolved in pure chloroform at room temperature to obtain a 3 wt% polymer solution.Aerwards, 1, 10 or 20 wt% of CORM-1 (based on PLA) was added and stirred under exclusion of light for 20 min yielding yellow homogeneous solutions.These mixtures have been electrospun at daylight (Fig. 1b).We nally obtained the loaded eece materials CORMA-1-PLA1, CORMA-1-PLA10, and CORMA-1-PLA20.These hybrid polymers are of yellowish color caused by the embedded CORM-1 (Fig. 1c).The color intensity is increasing from 1 wt% to 20 wt% loading.Attempts to prepare lms from casting solutions of PLA, PMMA and PS with CORM-1 as control samples failed due to phase separation, crystallization and nally decomposition of Mn 2 (CO) 10 during lm formation.This emphasizes even more that electrospinning is an ideal process for preparing CORM-containing hybrid materials.SEM images of electrospun non-wovens were recorded to determine the inuence of CORM-1 on the ber structure of PLA during incorporation (Fig. 2a-d).All non-wovens display roughly the same mean ber diameter of about 1 mm.The content of CORM-1 did not signicantly inuence the ber diameters.Interestingly, the images show porous nanobers (Fig. 2a-c and S1 †).In contrast, a control sample of CORMA-1-PLA20 (electrospun under exclusion of daylight) exhibited no comparable porosity (Fig. 2d).PLA bers without CORM-1 displayed a smooth morphology (Fig. S2c †). 29The number of uniformly distributed pores increases from CORMA-1-PLA1 to CORMA-1-PLA20.It is important to note that already 1 wt% of Mn 2 (CO) 10 is enough to produce a porous morphology in PLA bers (Fig. 2a).In addition, we performed Kr-BET and Hg porosimetry measurements on the CORMA-1-PLA non-wovens.Surface areas of 1-3 m 2 g À1 from a control PLA non-woven, CORMA-1-PLA10, and CORMA-1-PLA20 have been determined (Table S1 †).These values can be expected from typical ber diameters of around 1 mm in electrospun non-wovens.Unfortunately, we were not able to corroborate the BET measurements of CORMA-1-PLA10 and CORMA-1-PLA20 with SEM pictures (see Table S1 †).However, light-induced CO release from Mn 2 (CO) 10 in the electrospinning process is necessary to form these porous hybrid materials (for reproduced batches and their SEM images, see Fig. S2a and b †).Until now, porous bers within non-wovens have been produced by phase-separation of polymer blends and solvent mixtures in the electrospinning process. 45By using a photoCORM, we found a novel method to generate reliable nanoporous bers by triggering it with visible light.
SEM-EDX elemental mapping was conducted on a CORMA-1-PLA20 non-woven to determine the distribution of CORM-1 throughout the bers.Fig. 2e shows cross-sectional SEM-EDX elemental maps of Si (from underground Si wafer), C, O and Mn (see also Fig. S3 †).The pictures reveal a homogeneous distribution of manganese, and therefore CORM-1, within the polymeric bers.In addition, DSC measurements of CORMA-1-PLA have been performed.The thermogram displayed the expected glass transition temperature T g of PLA at 59 C and two exothermic conversions of Mn 2 (CO) 10 at 137 and 174 C (Fig. S7 †).The content of the carbonyl complex before thermal degradation and ber porosity did not have any signicant inuence on the T g of the carrier polymer. 29V-Vis and IR analyses were used to examine the identity of the incorporated manganese complex.UV-Vis spectra from the CORM-1 itself and the dissolved hybrid non-woven CORMA-1-PLA20 showed a comparable absorption band at 343 nm and a shoulder at 395 nm for both samples (Fig. S4 †).Thus, CORM-1 was substantially retained during high-voltage electrospinning.The actual amount of incorporated manganese carbonyl was also estimated.Samples of CORMA-1-PLA20 were dissolved in deaerated CHCl 3 and UV-Vis spectra were immediately recorded.The obtained absorbance was compared to the absorbance of a solution of pure CORM-1, its concentration representing the theoretical amount of 20 wt% within the matrix sample.We found that the absorption of the material samples corresponds to 56-59% of the initial CORM-1 concentration within the polymer (Fig. S4 †).For CORMA-1-PLA10 we obtained similar % values.These results were combined with another UV-Vis experiment, where CORM-1 was le standing in a non-deaerated CHCl 3 solution (analogue to the solution applied for electrospinning) in the dark.A decrease of the CORM-1 absorption band at 343 nm was monitored within 2 hours (Fig. S5 †).Therefore, we assume that the porous ber morphology originates from decomposition (and concomitant CO-gas generation) of CORM-1 in the chloroform solution during electrospinning by the inuence of daylight and air.
To determine the amount of incorporated Mn 2 (CO) 10 , we dissolved batches of CORMA-1-PLA10 and CORMA-1-PLA20 in aqueous nitric acid and measured the manganese content by ICP-MS analysis.For samples where 10 wt% of CORM-1 was originally embedded into PLA, a Mn amount was found that represents 7.9 wt% (s ¼ 0.3 wt%) of containing CORM-1.For CORMA-1-PLA20 the measured Mn content was 14.8 wt% (s ¼ 0.3 wt%) CORM-1 (see Table S2 †).Comparing UV-Vis and ICP-MS, we measured incorporation efficiencies from 56% to 79% of CORM-1 in PLA nanobers via the electrospinning process.
The structure of the incorporated CORM-1 was examined by infrared spectroscopy.We measured a spectrum of pure PLA bers (Fig. S6 †) and then subtracted it from spectra of the CORMA-1-PLA materials.The resulting difference ATR-IR spectra show the appearance of three n(C^O) vibration bands at 2046, 2031 and 2004 cm À1 , whereas n(Mn-CO) vibrations are found at 645 and 465 cm À1 next to the PLA bands aer incorporation of CORM-1 (Fig. 2f).Those bands are comparable to pure CORM-1 (Fig. S6 †).However, in CORMA-1-PLA1 the content of Mn 2 (CO) 10 is too small to be detected by ATR-IR and the porous morphology of CORMA-1-PLA1 displays also the CO loss from the Mn carbonyl (Fig. 2a).The IR results from CORMA-1-PLA10 and CORMA-1-PLA20 indicate that the major manganese carbonyl species retained is CORM-1.

CO release from CORMA-1-PLA
Light induced release of CO bubbles from CORMA-1-PLA20 with its high surface area (see Fig. 2c) was directly observed with a laser scanning microscope (LSM).The non-woven was covered with a cover lid to retain the CO gas.The formation of gas bubbles was observed within 25 seconds during illumination with the internal LSM lamp (405 nm).Fig. 3d shows the sample before illumination and aer 50 seconds of light exposure (for a video of the CO release see ESI †).Aer exhaustive illumination of the CORMA-1-PLA samples they turn dark yellow to brown.Manganese(IV) oxide (MnO 2 ) was qualitatively identied from an acidic CORMA solution with hydrochloric acid and iodine water.
The photo-induced loss of CO as the origin of the formed bubbles was conrmed by IR spectroscopy.A sample of CORMA-1-PLA20 was irradiated at 365 nm for 60 minutes and an ATR-IR spectrum was subsequently taken and depicted as the difference spectrum (Fig. 3e).The comparison between the irradiated and the non-irradiated sample of CORM-1-PLA20 shows a loss of the CO vibration bands at 2046, 2031, 2004, 645 and 465 cm À1 aer illumination (Fig. 3e, bands downwards).In contrast, no change in the PLA vibration bands can be observed (Fig. 3e, bands upwards).
CO detection under solvent free conditions was performed using a portable CO detector (Draeger Pac7000). 32CO release from CORMA-1-PLA20 samples was achieved by irradiation at three different wavelengths.Irradiation with a dental LED lamp at 440-480 nm (Translux lamp) allowed rapid application for CO release (Fig. S8b †).The CO release was tested in a closed vessel with xed duration of illumination (10 seconds ON) and 6 minutes OFF to reach equilibrium (see Fig. S8a † for the experimental setup).Fig. 3a shows that CO release is exclusively achieved through illumination, while no further CO is formed when the light source is shut off.Illumination at 365 nm and 480 nm was used to demonstrate CO release ability at UV-A and at wavelengths of visible light (see Fig. S9 † for the experimental setup). 23,24,50Comparison of these two wavelengths showed the energy dependency of the CO release mechanism.Both wavelengths are xed at the same light intensity (10 mW cm À2 ), but the CO release occurs more rapidly at 365 nm compared to 480 nm.This was shown by the calculated t 1/2 values of 309 AE 51 and 1289 AE 16 seconds (Fig. 3b). 50These data impressively reveal how our CORMAs can be tuned in terms of their CO release kinetics.Simply varying the wavelength of irradiation allows a difference of the CO release rate by a factor of four.
The quantication of the formed CO amount was accomplished via the gas IR technique.An IR gas cuvette was equipped with the non-woven sample (CORMA-1-PLA-20) and irradiated from the outside through a quartz window (Fig. S12 †).Repeated measurements resulted in an average value of 3.4 AE 0.3 mmol CO per mg sample (Fig. 3c).
CORMA-1-PLA10 was applied to a heterogeneous myoglobin assay in aqueous solution.When attached to a paperclip and introduced in a sealable uorescence cuvette, the non-woven can be irradiated from one direction while absorbance measurements can be applied in the orthogonal direction (Fig. S10 †).CO is released during irradiation at 365 nm (and 480 nm) and reacts with reduced horse heart myoglobin (Mb) under oxygen free conditions to form carboxy-myoglobin (Mb-CO, Fig. 4a).We quantied the rate of CO release by a half-life value. 51It has recently been shown by Poole et al. that watersoluble CORMs exhibit variable CO release rates dependent on the amount of sodium dithionite (reducing agent for Mb) in the myoglobin assay. 52It was suggested that sodium dithionite can directly react with a CORM and enhance the CO release rate.Therefore, we checked the effect of three different amounts of the reducing agent compared to the initial Mb in the heterogeneous assay (Fig. S11 †).Using 2, 20 and 200 equivalents of sodium dithionite we found release half-lives of 965 AE 63, 1305 AE 290, and 1180 AE 154 seconds, respectively.These data show that the given Mb-CO concentrations are formed in comparable time intervals.Thus, the amount of sodium dithionite does not signicantly inuence the rate of Mb conversion (Fig. S11 †).An additional myoglobin experiment was performed during 480 nm irradiation.The results in Fig. 4b show that Mb conversions occur much slower at 480 nm compared to 365 nm illumination, which conrms the wavelength dependency of the CO release. 23,24,50aching of CORM-1 from CORMA-1 ICP-MS was used to determine the loss of CORM-1 from CORMA-1-PLA10 and CORMA-1-PLA20 in an aqueous environment.In a typical experiment samples were soaked in water and agitated for 3 hours or 3 days.The amount of manganese in the aqueous supernatant was measured by ICP-MS.The amount  S2 in the ESI †).We found that around 0.5% of CORM-1 is lost from CORMA-1-PLA10, while CORMA-1-PLA20 only leaches approximately 0.1% aer 3 hours of soaking.Aer three days the amounts of loss are less than 0.8% for CORMA-1-PLA10 and less than 0.3% for CORMA-1-PLA20.We also checked whether the extent of leaching is inuenced during and aer irradiation with light.The samples were irradiated at 365 nm during agitation for one hour.Then the samples were removed and soaked in a fresh aqueous solution to determine leaching of the degradation products.It was found that during irradiation up to 2.4% of CORM-1 is lost.However, agitation aer irradiation showed again a lower leaching of the photolytic products of 0.8% maximum.These results show that water insoluble CORM-1 and its metal fragment photoproduct(s) are very efficiently retained by the nanoporous PLA bers.

Cell experiments and (photo)cytoxicity
To ensure that CORMA-1-PLA materials do not cause toxic effects in a biological environment before CO is released, the cytoxicity on 3T3 mouse broblast cells was determined in the dark.Criteria of evidence for cytotoxicity will be given: due to the 3-dimensional ber frameworks loss of detached dead cells could occur.Therefore, cytoxicity was not only determined by accurate quantities of dead cells but also by analyzing the changes in cell morphology and rough quantities that reveal non-toxic (below $5% of dead cells) and toxic effects (above 5%).The incorporation of different amounts of CORM-1 to the PLA did not signicantly inuence the cell morphology compared to a control sample (see Fig. S13 †).Aer 1 and 4 days of cell incubation the cells were still adherent and only a small amount of cells appeared more spherical than the control cells.Less than 5% of dead cells were found.Therefore, the samples were categorized as non-toxic.The cell number clearly increased and a dense cell population was found aer the incubation intervals (not shown) so that the proliferation of the cells was nearly not affected (Fig. S13 †).In contrast, phototoxicity was observed during illumination of the samples at 365 nm (surveyed aer 0, 15, and 60 minutes of irradiation; Fig. 5a).Samples of CORMA-1-PLA10 and CORMA-1-PLA20 were used to test the cell response during exposure to high concentrations of CO.A direct correlation between ongoing irradiation and toxic effects on cells was found for CORMA-1-PLA10 (Fig. 5b) and CORMA-1-PLA20 (Fig. S14 †).For both samples the amount of dead cells increased during illumination; the increase was more pronounced for CORMA-1-PLA20 (Fig. S14 †).Both experiments led to a change in cell morphology towards spherically shaped cells.These results indicate a strong phototoxic effect of the incorporated CORM-1 on 3T3 mouse broblasts compared to a pure PLA control sample.The contribution of UV-A light itself to the phototoxicity was negligible (see PLA control in Fig. 5b).
To evince that truly CO is responsible for the observed cell death, the phototoxicity experiments at 365 nm were repeated with a different setup.Instead of keeping cells and samples in the same well the non-woven sample was stored in one well and the cells were cultivated in the neighboring one, while a third control well was protected from CO intrusion (Fig. 5c).A cover lid over the whole well-plate avoided the loss of released CO into the atmosphere and allowed the diffusion of CO from the sample to the cells.Images in Fig. 5d show that more spherical and dead cells were found in the cell samples subjected to CO compared to the blank sample demonstrating the toxic effect of the pure CO gas on 3T3 mouse broblasts.It has to be noted here that the toxic effect of CO aer 60 minutes of illumination was lower when the material and cells were separated instead of being stored together during irradiation.The reason for this difference can be found in the CO diffusion between two wells.

C Conclusion
Embedding the photosensitive dimanganese decacarbonyl (CORM-1) into nanoporous brous non-wovens of polylactic acid has been achieved in this work.The manganese carbonyl is of dual advantage.In the electrospinning process at ambient light slight decomposition of the complex via the loss of CO induces a nanoporous structure in the bers.The resulting high surface area facilitates the interaction with light to generate an efficient triggerable CO delivery platform.In addition to the CO electrode and IR cuvette assay, a heterogeneous myoglobin assay was developed to characterize the light-induced CO release.Cytotoxicity of the biocompatible CORMAs on mouse broblast cells was very low in the dark.Exposure of the hybrid polymers to UV-A and visible light resulted in a rapid release of CO bubbles while the photoproducts were retained in the polymer matrix.Leaching of CORM-1 from the polymer matrix was negligible under physiological conditions due to the very low solubility of CORM-1 in water.The CO delivery platform has been tested by light-induced eradication of grown mouse broblast cells on the non-wovens.
The efficacy was dependent on the content of dimanganese decacarbonyl.The versatility of the presented CORMAs in future applications is outstanding.The material can be wrapped around biological targets (skin, etc.) or attached to optical bers.Currently we are investigating the non-wovens as a triggerable antibacterial platform 53 and as a possible CO storage system for onboard calibration of atmospheric CO sensors.

Materials
Chemicals and solvents were obtained from Fluka and VWR and used as received.Crystalline Mn 2 (CO) 10 (CORM-1) was bought from ABCR GmbH & Co. KG (Germany).Horse heart myoglobin was obtained from Sigma-Aldrich.Poly(L-lactide-co-D/L-lactide) 70/30 (PLA, Resomer LR 708, Boehringer Ingelheim Pharma GmbH & Co. KG, Germany) was used for electrospinning.A weight-average molar mass of 1.3 Â 10 6 g mol À1 was determined for the polymer by size exclusion chromatography using CHCl 3 as the solvent and polystyrene as the external standard.

Generation of CORMA-1-PLA non-wovens
The electrospinning process was carried out using the electrospinning apparatus E-Spintronic (Erich Huber GmbH, Germany).A stainless-steel straight-end hollow needle (0.4 mm) was used as the nozzle.A glass mirror of 3 mm thickness (30 Â 30 cm) was used as a collector plate for collecting the electrospun non-woven bers.The distance between the needle tip and the foil was maintained at 20 cm.The voltage was adjusted to 25 kV.A 3 wt% solution of the PLA in CHCl 3 containing 1, 10, and 20 wt % of CORM-1 related to the applied polymer was employed (CORMA-1-PLA1, CORMA-1-PLA10, and CORMA-1-PLA20).The polymer solution was fed at a constant rate of 1.5 ml h À1 through the syringe to the needle tip in the dark, resulting in the formation of bers with diameters of about 500 nm to 3 mm.The dimension of the obtained electrospun non-wovens was approximately 30 cm 2 with white (CORMA-1-PLA1), slightly yellow (CORMA-1-PLA10), and yellow (CORMA-1-PLA20) color, depending on the content of Mn 2 (CO) 10 . 31 Analysis of the manganese content of CORMA-1-PLA bers The overall manganese content within CORMA-1-PLA samples was determined aer sample destruction in 65% ultrapure nitric acid and subsequent dilution with MilliQ water.The quantity of manganese in the aqueous solutions was determined by inductively coupled plasma-mass spectrometry (ICP-MS) on an XSer-iesII from Thermo Fisher Scientic, Germany (isotope 55 Mn).
Calibration was performed with a multielement solution containing manganese ICP standard from Merck KGaA, Germany (CertiPUR).Measurements were performed against 101 Ru as the internal standard (c ¼ 20 mg l À1 in all samples).

Leaching of CORM-1 from CORMA-1-PLA bers
To determine the loss of CORM-1 from CORMA-1-PLA within an aqueous environment the non-woven samples were agitated in MilliQ water for three hours or three days (with or without previous 365 nm illumination) and the solution was subsequently mixed with nitric acid.The quantity of manganese in the aqueous solutions was determined by ICP-MS.

Spectroscopic characterization of CORM-1 and CORMA
Absorption spectra of the materials were recorded on an Analytik Jena Specord S 600 UV-Vis spectrometer.IR spectra were recorded in a range of 400-4000 cm À1 on an Equinox 55 FTIR/ FTNIR Spectrometer from Bruker Optik GmbH, Germany.

Other measurements
Scanning electron microscopy (SEM) was performed with a eld-emission scanning electron microscope Supra 55VP (Carl Zeiss AG, Germany).A Si wafer was used as the substrate material for the bers and Au was sputtered on the specimens to ensure sufficient electric conductivity for the analysis.The images were taken with an InLens-Detector using 5 keV excitation energy.Energy dispersive X-ray spectroscopy (EDX) was investigated using the SEM equipped with an EDX-system (Quantax with Si(Li)-detector, Bruker Nano GmbH, Germany).The non-woven mats were coated with evaporated carbon.For the measurements, an excitation energy of 2 keV was used.Spectra were recorded from crossed ber regions of the non-woven mat to get a larger excitation volume for EDX.
For optical investigation of CO release the laser scanning microscope LSM 700 (Carl Zeiss Microscopy GmbH, Germany) was applied.The non-woven mat (CORMA-1-PLA-20) was placed on an object slide, moistened with PBS buffer solution (pH ¼ 7.4) and covered with a cover slip in the dark.Aer adjustment the sample was irradiated with light of 405 nm of the integrated light emitting diode.Every 2 s an image was collected for over 50 s.

CO release prole of CORMAs
For heterogeneous myoglobin assays a given amount of CORMA-1-PLA10 was accurately weighed and xed on a paper clip.The clip and a small stirring bar were introduced into a uorescence cuvette that was sealed with a rubber septum.Horse heart myoglobin was dissolved in 2.5 ml phosphate buffer (pH 7.4), ltered and added to the cuvette.Nitrogen gas was bubbled gently through the solution for 5 minutes.Sodium dithionite was weighed into a Schlenk ask, dissolved in degassed phosphate buffer and 2, 20 or 200 equivalents compared to met-myoglobin were added to the cuvette.The non-woven was irradiated at 365 nm (or 480 nm) in a xed distance of 10 cm (1.4 mW cm À2 ) and UV-Vis absorption spectra were recorded in 5 minute intervals.The absorptions were recalculated to the corresponding Mb-CO concentrations and release half-life values were determined for quantication that is dened by the time taken for a solution of CORM (c ¼ x mM) to produce a solution of carboxy-myoglobin with c ¼ x/2 mM. 51ccordingly, the t 1/2 release value was used, where CORM-1 (resembling 20 mM if it was dissolved) produces 10 mM of Mb-CO.LED excitation was performed with a HD-LED module consisting of 5 vertically aligned LED lamps (INNOTAS Elektronik GmbH, Germany) in a sealable illumination box.The intensity of the lamps was determined with the Photodiode Power Sensor S120VC equipped with the USB Power and Energy Meter Interface PM100USB from Thorlabs GmbH, Germany.
CO release from CORMA in air was monitored with a portable CO detector from Draeger Safety Austria GmbH, Austria (Draeger Pac7000).CO release at 440-480 nm was achieved with a dental LED polymerization lamp (Translux Power Blue, Heraeus Holding GmbH, Germany).Further experiments at 365 and 480 nm were performed using the HD-LED modules applied in the myoglobin assay.The lamp intensities were adjusted to 10 mW cm À2 .
Quantication of CO release in air was achieved via gas IR spectroscopy within a self-made IR gas cuvette.A Varian 670-IR FTIR spectrometer (Agilent Technologies Deutschland GmbH, Germany) was used to detect CO.Four UV LEDs (365 nm; Nitride Semiconductors Co., Ltd., Japan) were used to irradiate the non-woven from outside the cuvette through the glass window during the IR measurement.The applied light power (1 mW cm À2 ) was determined with a FieldMaxII laser power meter (Coherent Inc., Germany).

Live/dead assay
Mouse broblast 3T3 cells were seeded onto the non-woven mats xed in cell crowns (Sigma-Aldrich, Germany) in daylight to a density of about about 25 000 cells cm À2 (50 000 cells cm À2 in (photo)cytotoxicity experiments) using DMEM culture medium (Biochrom-Seromed KG, Germany) supplemented by 10% fetal bovine serum.For different experiments on cytocompatibility and on (photo)cytotoxicity (see below) cells were stained on viability with 15 mg ml À1 uorescein diacetate and 1 : 10 000 diluted GelRed® stock solution (VWR International, Germany).An Axiovert 25 microscope (Carl Zeiss Jena GmbH, Germany) with Zeiss lter sets 44 (excitation: BP 475/40, beamsplitter: FT 500, emission: BP 530/50) and 14 (excitation: BP 510-560, beamsplitter: FT 580, emission: LP 590) and a halogen lamp were used to monitor green and red uorescence.Photomicrographs were recorded immediately aer staining using a CCD uor microscope imager MP 5000 (Intas Science Imaging Instruments GmbH, Germany).Imaging was supported by Image-Pro Plus soware (Media Cybernetics Inc., USA).The total number of cells and the percentage of living cells were calculated aer counting red-uorescent nuclei of dead cells and green-uorescent nuclei of living cells.

Cytocompatibility of CORMAs
To assess cytocompatibility under prevention of CO release, the non-woven mats CORMA-1-PLA1, CORMA-1-PLA10, CORMA-1-PLA20 and a PLA blank were incubated with the 3T3 broblast cell line in the dark for one and four days.

(Photo)cytotoxicity of CORMAs
To investigate the behavior of cells under CO release from the bers, the non-woven mats CORMA-1-PLA10, CORMA-1-PLA20 and a PLA control (blank sample spun from an acetone solution) were incubated with the 3T3 broblast cell line in the dark for one day.For induction of CO release materials were irradiated for 15 min as well as 1 h with UV light using an UV transilluminator (365 nm, 4 mW cm À2 ) within a dark box (Gel Imager, Intas Science Imaging Instruments GmbH, Germany).Aerwards the cells were cultivated for further 60 min and then stained with uorescein diacetate and GelRed®.In a control experiment seeded cells and the test material were stored in different adjacent wells and covered under one lid to examine the inuence of CO gassing from the sample wells into the cell wells.Blank cells were seeded in another well row and covered with foil to avoid CO gas inuences on the cells.The well plate was then incubated at 37 C for one day and the non-wovens were subsequently irradiated at 365 nm.

Fig. 1 Journal
Fig. 1 (a) Concept of CORM incorporation into a polymeric non-woven (resulting in a hybrid CORMA) to achieve water compatibility and retain the metal fragment (inactive CORM, iCORM) from the biological medium; (b) electrospinning of a dissolved mixture of polymer and CORM to produce the hybrid non-woven; (c) a sample of electrospun hybrid material CORMA-1-PLA1 (m z 0.6 mg).

Fig. 3 5 Paper
Fig. 3 (a) CO release profile of CORMA-1-PLA20 under exposure to 440-480 nm light (dentist lamp).Irradiation was accomplished in a sealed beaker (Fig. S8a †) in 10 second intervals (CO release) followed by an equilibration time of 6 minutes; (b) irradiation of CORMA-1-PLA20 at 365 nm (blue) and 480 nm (red) at a similar intensity of 10 mW cm À2 shows the wavelength dependency of the CO release rate; (c) total amount of CO released into the gas phase during irradiation at 365 nm determined via gas phase IR.Data points are means with a 95% confidence interval (inset: the rotationally resolved CO vibration band of measured IR spectra at different time intervals); (d) LSM pictures of light-triggered CO release from CORMA-1-PLA20: the sample before (left) and after illumination (right) at 405 nm in the microscope.The formed CO bubbles are shown in black patches; (e) difference spectra (the CORMA-1-PLA20 spectrum subtracted from the pure PLA spectrum) indicate the loss of CO vibration bands after irradiation at 365 nm.The PLA stays intact during UV-illumination.

Fig. 4 Journal
Fig. 4 CORMA-1-PLA10 in a heterogeneous myoglobin assay; (a) CO is released during irradiation and reacts with reduced horse heart myoglobin (Mb) under oxygen free conditions to form carboxy-myoglobin (Mb-CO); (b) irradiation wavelengths (365 nm in blue or 480 nm in red) influence the rate of CO release and myoglobin conversion.

Fig. 5 7 Paper
Fig. 5 Phototoxicity experiments with 3T3 mouse fibroblast cells; (a) experimental setup where cells and control (left part) or sample (right part) have been illuminated within the same well; (b) microscopic images of PLA control (60 min at 365 nm) and CORMA1-PLA-10 after 0, 15 and 60 minutes of exposure to UV-A light; (c) experimental setup with separated cells and samples.The released CO gas from the sample can diffuse to uncovered cells (right part), while control cells are protected from CO (left part); (d) microscopic images of a protected control and an unprotected cell sample after exposure of CORMA1-PLA-20 to UV-A light for 60 minutes.